P1458: MITAPIVAT AMELIORATES IN VITRO HUMAN Β THALASSEMIC ERYTHROID MATURATION INDEX AND MODULATES THE EXPRESSION OF THE CYTOPROTECTOR PEROXIREDOXIN-2.
Authors: Alessandro Matte’, Richard Pozzetto Huot, Andrea Mattarei, Alberto Ongaro, Alice Passarini, Angela Siciliano, Roberta Russo, Achille Iolascon, Carlo Brugnara, Lucia De Franceschi
Journal: HemaSphere (2023)

Topic: 27. Thalassemias Background: b thalassemia (b thal) is a worldwide hereditary red blood cell (RBC) disorder with limited therapeutic tools except for standard treatment with chronic RBC transfusions and iron chelation. In b thal, pathophysiologic studies have shown that oxidation plays a key role in both ineffective erythropoiesis and reduced survival of mature red cells into the peripheral circulation (Matte et al ARS 28: 1-14, 2018; Matte A et al. ARS 23: 1284, 2015). Up-regulation of cytoprotective systems such as peroxiredoxin-2 (Prx2) has also been reported (Matte A et al. ARS 23: 1284, 2015). Recently, in a mouse model of b thal, we showed that the pyruvate kinase (PK) activator mitapivat significantly improved anemia by targeting both ineffective erythropoiesis and hemolysis (Matte et al. JCI 131: e144206, 2021). We also found that mitapivat ameliorated the erythroid cell maturation index for in vitro CD34+ derived erythroblasts from b thal (codb039) patients (Matte et al. JCI 131: e144206, 2021). The results of phase 2 proof-of concept study (NCT03692052) in non-transfusion-dependent thalassemic patients support the beneficial effects of mitapivat on b thal (Kuo KHM et al Lancet 400: 493-501, 2022). Aims: Here, we asked whether both PKLR and PKM2 are expressed in human b thal erythroblasts and whether activation via mitapivat might affect the expression of peroxiredoxin-2 (Prx2) (Matte et al ARS 28: 1-14, 2018). Methods: We studied CD34+ derived erythroblasts from either healthy controls or b thal (codb039) patients (n=5). Mitapivat (2uM) was added to the cell cultures as previously described (Matte et al. JCI 131: e144206, 2021). PKM2 and PKLR expression was evaluated by immune-blot analysis using specific anti-PKM2 and -PKLR antibodies in erythroid precursors. Prx2 expression was evaluated by RT-PCR analysis. Results: The expression of PKLR declined during erythroid maturation in both heathy and b-thal erythroblasts. In contrast, PKM2 expression was significantly higher in erythroblasts from b-thal patients (cod039) compared to healthy cells, which display an almost undetectable PKM2 in the late phase of erythropoiesis. This suggests that an increased PKM2 may compensate for the PKR decline to ensure cell maturation in the presence of oxidative stress. This is of interest since mitapivat was previously shown to activate both PKM2 and PKLR isoforms (Kung C et al Blood 2017). In in vitro b-thal erythropoiesis, we confirmed that mitapivat significantly increased erythroid maturation as supported by increased late-erythroblasts at 11 days of cell culture, corresponding to the late phase of erythropoiesis (Matte et al. JCI 131: e144206, 2021). This was associated with down-regulation of Prx2, which we previously showed to be increased in b-thal erythroblasts in response to oxidative stress (Matte et al. ARS 23: 1284, 2015). Summary/Conclusion: Our data show that both PKLR and PKM2 isoforms are expressed in the late phase of b-thal erythropoiesis when compared to healthy controls. The ability of mitapivat to activate both PKM2 and PKLR might represent an added value to limit oxidation during b-thal erythropoiesis, as suggested by the down-regulation of Prx2 expression. Keywords: Erythropoieisis, beta thalassemia, Human CD34+ cell